Historical Mechanisms Driving the Evolution of Ligand Specificity in Steroid Hormone Nuclear Receptors Public
Colucci, Jennifer (2014)
Abstract
The genetic and biophysical mechanisms by which new
protein functions evolve are central concerns in evolutionary
biology and molecular evolution. Despite much speculation, we know
little about how protein function evolves in natural proteins.
Here, we use ancestral protein reconstruction (APR) to trace the
evolutionary history of ligand recognition in steroid hormone
nuclear receptors (SRs), an ancient family of ligand-regulated
transcription factors that enable long-range cellular communication
central to multicellular life. We found that the most ancestral SR,
ancSR1, was regulated by estrogens (steroids with aromatic A rings
and small substituents at their carbon 17 position). After a gene
duplication event, the duplicate SR, ancSR2, evolved specificity
towards progestagens and corticosteroids (nonaromatic
3-ketosteroids with bulky substituents at their carbon 17 position)
while excluding estrogens from the binding pocket. We show that
this switch from ancSR1- to ancSR2-specificity is mediated by the
evolution of several large-effect substitutions within the ligand
binding pocket (LBP) that confer a stable hydrogen-bond network for
the A ring of nonaromatic 3-ketosteroids. We show that recognition
of the hormone's carbon 17 substituent in ancSR2 was conferred via
a series of epistatic interactions that served to reposition the
ligand and exploit available hydrogen bond capabilities within the
ligand binding pocket. Finally, we show that ancestral receptors
can be modulated by modern pharmaceuticals and suggest that the SR
antagonist mifepristone may act as receptor modulator at the
proteins coactivator binding cleft.
Table of Contents
Distribution Agreement.. 1
ABBREVIATIONS. 13
ABSTRACT.. 16
By Jennifer Katherine Colucci. 16
CHAPTER 1: INTRODUCTION.. 18
Steroid Receptors regulate normal and disease physiologies. 20
Nuclear Receptor structure. 23
SR mechanism of activation. 24
Molecular Evolution. 26
Ancestral Gene Resurrection. 27
Figure 1.3: Nuclear Receptor Domain Architecture. 33
Figure 1.6: Model of the mechanism of NR activation. 36
A. Percent identity of SR LBDs: 38
References. 39
CHAPTER 2: EVOLUTION OF MINIMAL SPECIFICITY AND PROMISCUITY IN STEROID HORMONE RECEPTORS. 47
Abstract. 49
Author Summary. 50
Introduction. 51
Results and Discussion. 54
Reconstruction and characterization of ancestral proteins. 54
Ancestral structure-activity criteria. 56
Minimal specificity in SR evolution. 57
An evolutionary explanation for SR-mediated endocrine disruption. 59
Structural causes of SR promiscuity. 60
Promiscuity, selection, and neutrality in the evolution of signaling. 61
Methods. 63
Phylogenetics and ancestral sequence reconstruction. 63
Reporter activation assays. 64
Alternative ancestral reconstructions. 65
Protein expression. 66
Crystallization and structural analysis. 67
Acknowledgements. 69
Figures. 70
Figure 2.1: Evolutionary expansion of the steroid receptors and their ligands. 70
Figure 2.2: Ligand-recognition rules of ancSR1 and ancSR2. 72
Figure 2.3: Evolution of minimal specificity. 74
Figure 2.4: Structural causes of minimal specificity. 76
Figure 2.5: Histogram of posterior probabilities for ancSR2. 78
Figure 2.6: Histogram of posterior probabilities for ancSR1. 79
Figure 2.7: Dose activation curves of ancSR1. 80
Figure 2.8: Dose activation curves of ancSR2. 81
Figure 2.9: The specificity of ancSR1 is robust to uncertainty in the reconstruction. 82
Figure 2.10 The specificity of ancSR2 is robust to uncertainty in the reconstruction. 83
Figure 2.11: Sensitivities of extant human receptors to an estrogen, androgen, progestagen, and corticosteroid. 84
Figure 2.12: Activation of the estrogen receptor ligand binding domains of two annelids and human ER α . 85
Figure 2.13: AncSR2 is not activated by the nonsteroidal ER agonists diethylstilbestrol and genistein and is not inhibited by ICI182870 and 4-hydroxytamoxifen. 86
Figure 2.14: ML steroid receptor phylogeny for ancSR2. 87
Figure 2.15: ML steroid receptor phylogeny for ancSR1. 88
Figure 2.16: Unreduced 184-taxon steroid receptor gene duplication phylogeny. 90
Figure 2.17: Omit maps of progesterone and 11-deoxycorticosterone. 91
Table 2.1: Reconstructed sequence of ancSR2. 92
Table 2.2: Reconstructed sequence of ancSR1. 93
Table 2.3: ancSR1 and ancSR2 percent similarities. 94
Table 2.4: CID numbers for synthetic and natural steroids used in this study. 95
Table 2.5: Fold preferences for hormone pairs. 96
Table 2.6: Data collection and refinement statistics. 97
Table 2.7: Receptors and organisms used for phylogenetic analyses. 98
Table 2.8: ancSR2 sequence comparison. 99
References. 100
CHAPTER 3: BIOPHYSICAL MECHANISMS FOR LARGE-EFFECT MUTATIONS IN THE EVOLUTION OF STEROID HORMONE RECEPTORS. 105
Introduction. 108
Protein biophysics and evolution. 108
An evolutionary shift in hormone specificity. 109
Results and Discussion. 111
Phylogenetic and structural analyses to identify causal mutations. 111
Two large-effect replacements shifted hormone specificity. 111
Structural mechanisms for the shift in specificity. 112
Changes in the energetic landscape of ligand binding. 113
Experimental analysis of changes in dynamics. 115
Arg82 is necessary for ligand-specificity. 116
Evolution of proteins as complex physical systems. 117
Methods. 119
Reporter activation assays. 119
Sequence conservation analysis. 120
Molecular dynamics methods. 121
Free energy landscapes. 122
Characterization of water-penetrated states. 124
HDX-MS. 124
Acknowledgements. 128
Figures. 129
Figure 3.1: Evolution of ancSR1 and ancSR2 specificity. 129
Figure 3.2: Large-effect historical mutations drove the evolution of new ligand specificity. 132
Figure 3.3: Two historical mutations altered the energetic landscape of protein-ligand binding. 133
Figure 3.4: Ligand-specific disruption of the A-ring hydrogen-bond network. 134
Figure 3.5: Cognate steroids of the six human steroid receptors. 136
Figure 3.6: A-ring ligand contacts are largely conserved between ancSR2 (magenta) and ancSR2 (blue). 137
Figure 3.7: Representative dose activation curves of ancSR2/Q41e/M75l and ancSR2 wild-type. 138
Figure 3.8: Representative dose activation curves of ancSR1 and ancSR1/e41Q/l75M. 139
Figure 3.9: A control MD simulation with the apo protein. 140
Figure 3.10: Derived amino acids introduce a new direct contact with the norP 3-keto group. 141
Figure 3.11 Populated rotamers of Glu41 and Gln41. 142
Figure 3.12: Dependence of number of states on ΔGbarrier. 143
Figure 3.13: Non-aromatized steroid with 3-hydroxyl does not populate frustrated hydrogen bond networks. 144
Figure 3.14: Historical mutations cause increased peptide solvent exchange in a ligand-dependent manner. 145
Figure 3.15: Model fits to incorporation vs. time data for the five peptides which exhibited decreased NPT-specific protection factors in the derived state. 147
Table 3.1: Pubmed compound identifier (CID) numbers for cholesterol and the synthetic and natural steroid hormones tested in this study. 148
Table 3.2: Conservation analysis of extant naSRs and ERs. 154
Table 3.3: Simulations display additivity: absolute free energies of barriers are the same for i → j versus j→i transitions. 155
Table 3.4: Transition matrices for top 95% of observed states with 2 kcal/mol energy cutoff. 156
Table 3.5: HDX-MS kinetics model selection. 157
References. 158
CHAPTER 4: X-RAY CRYSTAL STRUCTURE OF THE ANCESTRAL 3-KETOSTEROID RECEPTOR - PROGESTERONE - MIFEPRISTONE COMPLEX SHOWS MIFEPRISTONE BOUND AT THE COACTIVATOR BINDING SURFACE.. 162
Abstract. 163
Introduction. 164
Materials and Methods. 166
Reagents. 166
Expression and Purification. 166
Crystallization, data collection, structure determination and refinement. 167
Reporter Gene Assays. 167
Results. 169
Overall Structure. 169
Mifepristone binds at two distinct surface sites. 170
Improved resolution of the ancSR2-progesterone structure permits visualization of D-ring contacts. 171
Discussion. 172
Acknowledgements. 176
Figures. 177
Figure 4.1: Crystals of the ancSR2-progesterone-mifepristone complex and in vitro activation data. 177
Figure 4.2: Overall structure of the ancSR2-progesterone-mifepristone complex. 178
Figure 4.3: Omit maps of bound ligands. 179
Figure 4.4: Mifepristone binding site interactions. 180
Figure 4.5: Mifepristone occupies the coactivator protein space. 181
Figure 4.6: Global alignment of progesterone-bound steroid receptors. 183
Figure 4.7: Mifepristone and 4-hydroxytamoxifen show similar binding modes to the steroid receptor coactivator binding cleft. 184
References. 185
CHAPTER 5: EXPRESSION, PURIFICATION, AND CRYSTALLIZATION OF THE ANCESTRAL ANDROGEN RECEPTOR-DHT COMPLEX.. 189
Abstract. 190
Introduction. 191
Materials and Methods. 193
Reagents. 193
Cloning. 193
Expression and Purification. 194
Crystallization and Data Collection. 194
Results and Discussion. 196
Acknowledgements. 197
Figures. 198
Figure 5.1: Following a series of affinity columns, ancAR1-DHT was purified to homogeneity. 198
Figure 5.2: Crystals of ancAR1-DHT. 199
Figure 5.3: Diffraction image of an ancAR1-DHT crystal. 200
Table 5.1: Data collection statistics for AncAR1-DHT-Tif2. 201
References. 202
CHAPTER 6: BEYOND MINIMAL SPECIFICITY: EVOLVING THE ABILITY TO DISCRIMINATE AMONG DIVERSE 3-KETOSTEROIDS. 211
Abstract. 212
Introduction. 213
Materials and Methods. 215
Reagents. 215
Structural Analysis. 216
Mutagenesis. 216
Reporter activation assays. 216
Results. 218
Comparison of estrogen versus progesterone recognition in the ligand binding pocket 218
Which amino acid substitutions facilitate recognition of bulky carbon 17 substituents?. 219
Discussion. 221
Figure 6.1: Phylogeny of the Steroid Receptor lineage. 223
Figure 6.2: Rotation of the 17-acetyl ligand in the binding pocket allows for exploitation of pre-existing hydrogen bond capacity. 224
Figure 6.3: Forward Evolution of D-ring residues increased preference for 17-acetyl ligands. 225
Figure 6.4: Epistatic interactions shaped ancSR2 evolution. 226
Figure 6.5: Evolutionary pathway to the evolution of 17-acetyl recognition. 227
Table 6.1: Hormone sensitivity of WT ancSR2 and mutants. 228
References. 229
CHAPTER 7: DISCUSSION.. 231
How did the differences in ligand specificity between the ERs and naSRs evolve? 232
What are the mechanisms that dictate the ligand preferences of ERs and naSRs? 233
How can ancestral proteins be used to understand modern pharmacology? 235
How do epistatic interactions influence the evolution of ligand specificity? 236
Composite discussion. 238
Future Directions. 240
Can we completely recapitulate the switch in hormone selectivity from ancSR1 to ancSR2? 240
What factors contribute to the evolution of androgen specificity? 241
References. 242
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